Unlike humans, plants do not have an immune system, but at different stages of evolution they have developed a wide array of defense mechanisms in order to withstand pathogenic challenges. A well-characterized mechanism of plant defense is mediated by interaction between a plant's disease-resistance proteins, encoded by disease-resistance (R) genes, and the corresponding avirulence (Avr) genes expressed by the pathogen. (Bent, A F and Mackey, D, 2007, Elicitors, Evectors, and R-genes: the new paradigm and a lifetime supply of questions, Annu Rev Phytopathol, 45:399-436.). This interaction leads to activation of the plant's response, which in turn results in increased resistance to disease. The putative ligand-receptor complex initiates a series of signal transduction cascades contributing to disease resistance. (Baker, B et al., 1997, Signaling in plant-microbe interactions, Science, 273:726-733). Some cellular events that characterize resistance include oxidative burst, induction of defense gene expression, and rapid cell death at the site of infection. (Dhalowal, H S and Uchimiya, H, 1999, Genetic engineering for disease and pest resistance in plants, Plant Biotechnol, 16:255-261).
There are various types of disease-resistance genes. During different evolutionary events, plants have gained and omitted different disease-resistance genes. Therefore, a plant that displays resistance against a certain pathogen may have the gene(s) necessary for resistance against that pathogen. Consequently, identification of disease-resistant genes is a promising field of plant molecular biotechnology.
Disease resistance (R) genes, which interact with the Avr genes of different pathogens, have been isolated from numerous plant species. These genes confer resistance to a wide range of plant pathogens, including bacteria, fungi, oomycetes, viruses, and nematodes. (Baker et al., 1997; Bent, 1996; Hammond-Kosack, K E and Jones, J D G, 1997, Plant disease resistance genes, Annual Review of Plant Physiology and Plant Molecular Biology, 48:573-605; Ellis, C N et al., 1988, Topical Tretinoin for Photoaged Skin-Reply, JAMA, 259(22):3274-78). Although the products of avirulence genes typically have little homology with each other, the reported R genes have conserved functional domains, such as the protein kinase (PK) domain, Leucine-rich regions (LRR), and nucleotide-binding sites (NBS). (Bent, 1996; Ellis et al., 1988).
Based on the structure of R gene products, the R genes can be classified into four main classes: Nucleotide-Binding Site-Leucine-Rich Repeat (NBS-LRR); Leucine-Rich Repeat-Trans-Membrane domain-Protein Kinase (LRR-TM-PK); Leucine-Rich Repeat-Trans-Membrane domain (LRR-TM); and Protein Kinase (PK). (Bent, 1996; Ellis et al., 1988; Hammond-Kosack and Jones, 1997). The largest number of characterized R proteins is of the NBS-LRR type. These NBS-LRR proteins can be further subdivided into two sub-families based on the presence or absence of a region homologous to the toll and interleukin-1 receptor (TIR) domain at their N-terminus. (Baker et al., 1997; Parker, M F et al., 1997, Molecular characterization of adenocarcinoma of the cervix, Gynecol Oncol., 64(2):242-51). The two sub-families are designated TIR and non-TIR, respectively. Most members of the non-TIR subfamily encode a putative coiled-coil (CC) domain at their N-terminus. (Pan, P et al., 2000, Determination of the in situ bactericidal activity of an essential oil mouth rinse using a vital stain method, J Clin Periodontol., 27:256-261). TIR and non-TIR NBS-LRR R genes can be distinguished by amino acid motifs found within the NBS domain itself. (Meyers, B C et al., 2003, Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis, Plant Cell, 15:809-834; Pan et al., 2000). They also differ at the functional level, based on their apparent involvement in different signal transduction pathways. (Aarts, N et al., 1998, Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis, Proc. Natl. Acad. Sci. USA, 95:10306-10311).
NBS sequences are abundant in plant genomes. (Meyers et al., 1999). In fact, the Arabidopsis genome contains 150 NBS-encoding genes, including 85 TIR NBS-LRR genes, 43 non-TIR NBS-LRR genes, and numerous truncated TIR or CC-NBS genes that lack an LRR domain (The Arabidopsis Genome Initiative, 2000). No precise functions have yet been established for each of these resistance gene analogs (RGAs). (Kanazin, V. et al., 1996, Resistance gene analogs are conserved and clustered in soybean, PNAS, USA, 93(21):11746-11750; Leister, D et al., 1996, PCR based approach for isolating pathogen resistance genes from potato with potential for wide application in plants, Nature Genetics, 14(4):421-429; Yu, Y G et al., 1996, Isolation of a super family of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site, PNAS, USA 93(21):11751-11756). Thus, RGAs are valuable as potential sources of active R genes.